title: Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.
abstract: Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.
authors.affiliation: Instituto de la Grasa (CSIC), Department of Biochemistry and Molecular Biology of Plant Products, 41012, Sevilla, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Aznar-Moreno
authors.forename: Mónica
authors.initials: M
authors.lastname: Venegas Calerón
authors.forename: Enrique
authors.initials: E
authors.lastname: Martínez-Force
authors.forename: Rafael
authors.initials: R
authors.lastname: Garcés
authors.forename: Robert
authors.initials: R
authors.lastname: Mullen
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Gidda
authors.forename: Joaquín J
authors.initials: JJ
authors.lastname: Salas
mesh_terms: D000595:Amino Acid Sequence; D003066:Coenzyme A Ligases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013229:Stearic Acids / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000166:cytology / Q000235:genetics; D014162:Transfection
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013229:Stearic Acids; D049452:Green Fluorescent Proteins; D019301:Oleic Acid; C031183:stearic acid; D003066:Coenzyme A Ligases